Molecular Methods For Diagnosis Research Articles

Pathology, immunohistochemistry and molecular study of cattle screened by comparative tuberculin test for diagnosis latent form of Johne’s disease

Mycobacterium avium subsp. paratuberculosis causes Johne’s disease in cattle and other ruminants. It is one of the most widespread in terms of prevalence and one of the most important diseases in the livestock industry in terms of economic importance. In the latent form of the disease, animals can act as carriers. The main purpose of this study was to evaluate avian tuberculin in a single intradermal comparative tuberculin test (SICTT), histopathology, immunohistochemistry, and molecular methods for diagnosis of infected cows with the latent form of the disease. Twenty-three asymptomatic cows and without post-mortem signs of the disease were selected using the single intradermal comparative tuberculin test and were tested.In this study, avian tuberculin in SICTT was able to in diagnosing the latent form of the disease. The results confirm immunohistochemistry and real-time PCR as a sensitive and reliable diagnostic complementary approach for detecting latent forms of Paratuberculosis.

DdPCR increases detection of SARS-CoV-2 RNA in patients with low viral loads

RT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the onset of the pandemic, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis method. Droplet digital PCR (ddPCR) is a third-generation PCR technique that is particularly adapted to detecting low-abundance targets. We developed two-color ddPCR assays for the detection of four different regions of SARS-CoV-2 RNA, including non-structural (IP4-RdRP, helicase) and structural (E, N) protein-encoding sequences. We observed that N or E subgenomic RNAs are generally more abundant than IP4 and helicase RNA sequences in cells infected in vitro, suggesting that detection of the N gene, coding for the most abundant subgenomic RNA of SARS-CoV-2, increases the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of Greater Paris. We found that 8.6% of informative samples (n = 16/185, P < 0.0001) initially scored as “non-positive” (undetermined or negative) by RT-qPCR were positive for SARS-CoV-2 RNA by ddPCR. Our work confirms that the use of ddPCR modestly, but significantly, increases the proportion of upper airway samples testing positive in the framework of first-line diagnosis of a French population.

Modern diagnostics of microsporia

Objective — to develop a method of modern molecular genetic diagnosis of microsporia in children based on polymerase chain reaction (PCR), which will allow identification of the pathogen of Microsporum canis at the DNA level.&#x0D; Materials and methods. The study included 40 patients with microsporia of smooth skin, scalp, scalp and smooth skin. The biological materials for the research were scales from the smooth skin and scalp, hair from the scalp of patients with microsporia. A study of 40 samples of biological material was carried out in patients with microsporia of smooth skin, microsporia of the scalp, microsporia of the scalp and smooth skin. At the first stage, DNA isolation of Microsporum canis was carried out. Then PCR was carried out to increase the copies of the DNA region using specific primers. The final step was typing 40 samples of clinical material of patients.&#x0D; Results and discussion. PCR diagnostics made it possible to identify the DNA of Microsporum canis in all 40 samples of biological material of patients with microsporia. In our study, we developed a PCR-based method for diagnosing microsporia, which uses a set of two MC primers (regions of the beta tubulin gene of Microsporum canis). For internal control of the course of amplification and the quality of biomaterial sampling, specific primers of APOE (a region of the human apolipoprotein E gene) were also used.&#x0D; Conclusions. In order to improve the precise specific diagnosis of microsporia in children, a method of modern molecular genetic diagnostics based on polymerase chain reaction (PCR) has been developed, which allows identification of the Microsporum canis pathogen at the DNA level. Analysis of the molecular structure of the genome of Microsporum canis proved that the most objective diagnosis of microorganisms is the PCR method. The developed method of DNA diagnostics based on PCR using specific primers can be included in the algorithm for detecting Microsporum canis in humans.

Safety and Tolerability of Mosquito Bite-Induced Controlled Human Infection with Plasmodium vivax in Malaria-Naive Study Participants-Clinical Profile and Utility of Molecular Diagnostic Methods.

Plasmodium vivax controlled human malaria infection (PvCHMI) is an important tool for evaluation of drugs, vaccines, and pathologies associated with this parasite. However, there are few data on safety due to limited numbers of PvCHMIs performed. We report clinical and laboratory data, including hematological and biochemical profiles and adverse events (AEs), following mosquito bite-induced PvCHMI in malaria-naive study participants. Malaria diagnosis and treatment initiation was based on microscopic analysis of Giemsa-stained slides. Exploratory molecular assays were used to detect parasites using real-time polymerase chain reaction (PCR). AEs were mild to moderate and no study-related severe AEs were observed in any study participants. The majority of symptoms were transient, resolving within 48 hours. Molecular diagnostic methods detected parasitemia in 100% of study participants before malaria diagnosis using microscopy. Of reported AEs, microscopy detected 67%-100%, quantitative PCR 79%-100%, and quantitative real-time reverse-transcription PCR 96%-100% of study participants prior to appearance of symptoms. Almost all symptoms appeared after initiation of treatment, likely as known consequence of drug treatment. PvCHMI is safe with the majority of infections being detected prior to appearance of clinical symptoms, which can be further alleviated by using sensitive molecular methods for clinical diagnosis. Clinical Trials Registration. NCT01157897.

CANINE VIRAL ENTERITIS – OLD AND NEW PATHOGENS

Canine viral eneritis is a frequent problem in clinical veterinary practice. From the discovery of canine parvovirus in the late 1970s to the present day, viruses have been one of the most common causes of diarrhea, especially in younger categories of dogs. The following primary causes of eneritis in dogs are most often isolated: parvovirus types 1 and 2, corona and rota virus. Modern molecular methods of diagnosis in the feces of dogs have proven the presence of new viruses, which lead to milder forms of enteritis (circoviruses, picornaviruses, astroviruses, calici and paramyxoviruses) in dogs. The presence of the virus in the feces of dogs with symptoms of diarrhea was proven in 40– 60% of the examined samples.

Argonaute with stepwise endonuclease activity promotes specific and multiplex nucleic acid detection

Argonaute proteins (Agos) from thermophiles function as endonucleases via guide-target base-pairing cleavage for host defense. Since guides play a key role in regulating the catalytic specificity of Agos, elucidating its underlying molecular mechanisms would promote the application of Agos in the medical sciences. Here, we reveal that an Ago from Pyrococcus furiosus (PfAgo) showed a stepwise endonuclease activity, which was demonstrated through a double-stranded DNA cleavage directed by a single guide DNA (gDNA) rather than a canonical pair of gDNAs. We validated that the cleavage products with 5'-phosphorylated ends can be used as a new guide to induce a new round of cleavage. Based on the reprogrammable capacity of Ago’s stepwise activity, we established a rapid and specific platform for unambiguous multiplex gene detection, termed Renewed-gDNA Assisted DNA cleavage by Argonaute (RADAR). Combined with a pre-amplification step, RADAR achieved sensitivity at the femtomolar level and specificity with at least a di-nucleotide resolution. Furthermore, RADAR simultaneously discriminated among multiple target sequences simply by corresponding multiple guides. We successfully distinguished four human papillomavirus serotypes from patient samples in a single reaction. Our technique, based on the unique properties of Ago, provides a versatile and sensitive method for molecular diagnosis.

A new molecular method for rapid etiological diagnosis of sepsis with improved performance.

The value of blood cultures for confirming the clinical diagnosis of sepsis is suboptimal. There is growing interest in the potential of real-time PCR technology by detection of minute amounts of pathogen DNA in patient blood samples with results available within 4-6h. Adopting a two-step approach, we evaluated the compliance of two versions of the MicrobScan assay on a total of 748 patients with suspected bloodstream infections. The results obtained with a second version of the MicrobScan assay are characterized by increased specificity (from 95.1to 98.2%) and sensitivity (from 76.7 to 85.1), increased throughput and the possibility of simultaneously testing different kinds of samples collected from the potential sites of infection and utilizing different syndromic panels.

Difficulties in the diagnosis of HbS/beta thalassemia: Really a mild disease?

BackgroundHbS/b cases having clinical, hematologic and electrophoretic similarities cannot be sufficiently distinguished from sickle cell anemia cases and are misdiagnosed as sickle cell anemia. This study will investigate the congruence between the HPLC thalassemia scanning tests and the laboratory findings compared to the DNA sequence analysis results of the patients diagnosed with SCA between 2016 and 2020. This study also aims to indicate the current status to accurately diagnose sickle cell anemia and HbS/b in the light of hematologic, electrophoretic and molecular studies.MethodsFourteen patients who were diagnosed with SCA in hospitals at different cities in Turkey and followed by the Thalassemia Diagnosis, Treatment and Research Center, Muğla Sıtkı Koçman University were included in this retrospective study. The socio-demographic characteristics, hemogram, hemoglobin variant analysis results and DNA chain analysis results of the patients were taken from the database of the centre and then examined. The informed consents were taken from the patients. The patients were administered a survey containing questions about transfusion history and diagnostic awareness. The Beta-Thalassemia mutations were analysed using a DNA sequencer (Dade Behring, Germany) based on the Sanger method.ResultsAccording to the DNA sequence analysis, the results of these patients diagnosed with SCA in hospitals in different cities of Turkey were the following: of 14 patients, 8 had HbS/b0, and HbS/b+ and one had HbS carrier, and one had Hb-O, and three had SCA. The patient with HbS carrier status also contains three additional mutations, all of which are heterozygous. We discovered that although two of three mutations, which are c.315+16G>C and c.316-185C>T, are previously reported as benign, at least one of the two mentioned mutations, when combined with HbS, causes transfusion-dependent HbS/b.ConclusionsBriefly, HbSS and HbS/b thalassemia genotypes cannot be definitely characterized by electrophoretic and hematologic data, resulting in misdiagnosis. c.315+16G>C and c.316-185C>T are previously reported as benign; at least one of the two mentioned mutations, when combined with HbS, causes transfusion-dependent HbS/b. In undeveloped or some developing countries, molecular diagnosis methods and genetic analyses cannot be used. If mutation analyses could be performed, then such differential diagnosis errors would reduce. However, if mutation analysis cannot be performed, other methods such as HPLC, capillary electrophoresis absolutely be sought to have insight into the parental carriage status.

Behcet-like familial autoinflammatory syndrome

Behcet-like familial autoinflammatory syndrome is a rare autoinflammatory disease with an autosomal dominant mode of inheritance, which is based on heterozygous mutations in the TNFAIP3 gene. It is characterized by recurrent fever, recurrent oral-genital ulcers. Currently, only few patients with this disease are described in the world literature. The clinical case presented in the article demonstrates a new mixed phenotype of pathology in the form of a combination of symptoms resembling Behcet’s disease and autoimmune lymphoproliferative syndrome. It allows us to enrich our knowledge about the genetic nature of autoinflammatory familial Behcet-like syndrome, and indicates the need to more active use of molecular genetic research methods in the differential diagnosis of these diseases.

The unprecedented epidemic-like scenario of dermatophytosis in India: II. Diagnostic methods and taxonomical aspects.

Trichophyton (T.) mentagrophytes now accounts for an overwhelming majority of clinical cases in India, a new "Indian genotype" (T. mentagrophytes ITS genotype VIII) having been isolated from skin samples obtained from cases across a wide geographical distribution in this country. The conventional diagnostic methods, like fungal culture, are, however, inadequate for diagnosing this agent. Thus, molecular methods of diagnosis are necessary for proper characterization of the causative agent. The shift in the predominant agent of dermatophytosis from T. rubrum to T. mentagrophytes, within a relatively short span of time, is without historic parallel. The apparent ease of transmission of a zoophilic fungus among human hosts can also be explained by means of mycological phenomena, like anthropization.

Large-Scale RT-qPCR Diagnostics for Seed Potato Certification

Every year, Agroscope examines nearly 300,000 tubers for the presence of viruses, as regulated for the certification of seed potatoes intended for Swiss growers. Since 2016, this examination has been performed via RT-qPCR on dormant tubers directly after harvest. This method offers fast results and eliminates the need for the use of Rindite, which is a toxic and polluting gaseous compound previously used in Switzerland to break the dormancy of seed tubers. The implementation of this molecular analytical method for the routine diagnosis of regulated viruses makes it possible to conduct additional analyses via Illumina sequencing to assess the conformity of the primers and probes used with the sequences of the different viral isolates. This form of quality control in routine diagnosis is a source of information that can answer more fundamental scientific questions related to the epidemiology of viral strains related to certification. The datasets produced in this framework can also be used to explore the diversity of rare or unknown virus species in potato crops.

Design and Development of Reverse Slot Blot for the Simultaneous Detection of Rare and Regional Specific Mutations in the Beta Globin Gene in Khuzestan Province of Iran.

Beta-thalassemia is the most frequent hemoglobin disorder in Iran resulting from disrupting mutations in the beta globin (HBB) gene that causes decreased or complete absent of beta-globin chains. The screening of beta-thalassemia minor and major individuals and prenatal diagnosis is important for familial planning. Therefore, it is essential, depending on the ethnicity and local frequency of changes, to develop a rapid and accurate method for molecular diagnosis of beta-thalassemia. Here, we developed reverse slot blot (RSB) assay for the simultaneous detection of six common pathogenic changes in the HBB gene (-88, -28, IVSII-745, IVSII-848, Codon 6 [G → A] for HbC, Codon 6 [A → T] for HbS) in the Khuzestan Province of Iran. We designed normal and mutant oligonucleotide probes for each selected mutation and fixed them on positively charged nylon membrane. In the next step, a multiplex-polymerase chain reaction (PCR) performed for the amplification of the entire HBB gene using labelled 5'-biotinylated primers. The PCR products were hybridized to immobilized oligonucleotide probes on the membrane at the appropriate temperature. Finally, we developed the membrane by chemically colorimetric reaction using nitro-blue tetrazolium-5-Bromo-4-chloro-3-indolyl phosphate. For the best probe concentration, we made a serial dilution of probe pairs for each mutation. The optimal probe concentration for each mutation varied from 25 to 50pmol. In the next step, DNA samples from homozygous affecting individuals were subjected for multiple PCR. Hybridization of each PCR products on the nylon membrane with probe pairs revealed specific bands with expected signal intensity without any background. Our designed RSB test is a rapid, sensitive and cost-effective method for screening of regional specific beta-thalassemia mutations in the Khuzestan population of Iran, which might be extended for the detection of any desired pathogenic changes.

The Association between Vaginal Dysbiosis and Reproductive Outcomes in Sub-Fertile Women Undergoing IVF-Treatment: A Systematic PRISMA Review and Meta-Analysis

Recent advances in molecular microbiology have enabled refined studies of the genital tract microbiota. This constitutes the basis of the present updated systematic review and meta-analysis which investigate vaginal dysbiosis (VD) as defined by either microscopy (e.g., Nugent score for bacterial vaginosis) or molecular methods (qPCR and Next Generation Sequencing) to evaluate the impact of VD on the reproductive outcomes in women undergoing IVF-treatment. A total of 17 studies were included, comprising 3543 patients and with a VD prevalence of 18% (95%CI 17–19). Across all methods, VD is a significant risk factor for early pregnancy loss in IVF (Relative risk (RR) = 1.71 95%CI 1.29–2.27). Moreover, a predefined sub-analysis of studies using molecular methods for VD diagnosis showed a significant reduction in the clinical pregnancy rate when compared to normal vaginal microbiota patients (RR = 0.55 95%CI 0.32–0.93). However, regardless of diagnostic methodology, VD did not significantly influence live birth rate (LBR). In conclusion, molecular tools have provided a more detailed insight into the vaginal microbiota, which may be the reason for the increased adverse effect estimates in IVF patients with molecularly defined VD. However, the quality of evidence was very low across all outcomes according to GRADE and thus, more studies are warranted to understand the impact of VD in IVF.

A review of diagnosis and treatment of syndromic hearing loss

Hereditary deafness is divided into syndromic hearing loss and non-syndromic hearing loss according to whether it is accompanied by other system dysfunction. The early identification and diagnosis of syndromic hearing loss is very important, including clinical and molecular diagnosis. Early diagnosis can predict the progress of hearing loss, other systemic disorders and guide treatment. Thus otolaryngologists are likely to become the first doctors to treat children with syndromic hearing loss, it is more necessary to master the clinical and molecular diagnosis methods of common syndromic hearing loss, and cooperate with doctors of other relevant departments for early intervention and treatment. Therefore, this article reviewed the common features, molecular diagnostic methods and treatment strategies for syndromic hearing loss.

Fantastic Downy Mildew Pathogens and How to Find Them: Advances in Detection and Diagnostics.

Downy mildews affect important crops and cause severe losses in production worldwide. Accurate identification and monitoring of these plant pathogens, especially at early stages of the disease, is fundamental in achieving effective disease control. The rapid development of molecular methods for diagnosis has provided more specific, fast, reliable, sensitive, and portable alternatives for plant pathogen detection and quantification than traditional approaches. In this review, we provide information on the use of molecular markers, serological techniques, and nucleic acid amplification technologies for downy mildew diagnosis, highlighting the benefits and disadvantages of the technologies and target selection. We emphasize the importance of incorporating information on pathogen variability in virulence and fungicide resistance for disease management and how the development and application of diagnostic assays based on standard and promising technologies, including high-throughput sequencing and genomics, are revolutionizing the development of species-specific assays suitable for in-field diagnosis. Our review provides an overview of molecular detection technologies and a practical guide for selecting the best approaches for diagnosis.

СУЧАСНІ МЕТОДИ ЕТІОЛОГІЧНОЇ ДІАГНОСТИКИ ГОСТРИХ НЕГОСПІТАЛЬНИХ ІНФЕКЦІЙ НИЖНІХ ДИХАЛЬНИХ ШЛЯХІВ

MODERN METHODS OF ETIOLOGICAL DIAGNOSING OF ACUTE COMMUNITY-ACQUIRED LOWER RESPIRATORY TRACT INFECTIONS O. L. Bororova, Y. O. Dziublyk, V.A. Iachnyk Abstract The review presents the possibilities presented by various methods of etiological diagnostics used in pulmonology. The main method of diagnosing acute community-aquired lower respiratory tract infections is the microbiological approach which includes microscopy of patient’s material with Gram staining, cultures on nutrient media, isolation of culture, identification and determination of susceptibility of a microorganism to antibiotics. But unfortunately the etiologocal factor cannot be detected in about half of patients. Recently, the popularity of molecular methods of etiological diagnosis has grown. They are characterized by greater sensitivity to microbiological methods and allow to get results faster. Molecular diagnostic tests are divided into four categories depending on the mechanism based on them: immunoassay, hybridization methods, amplification and sequencing methods. Among the tests based on the principles of immunoassay, noteworthy are rapid tests, which are most consistent with the idea of an ideal diagnostic tool in the field of laboratory medicine. They are fast, simple, cheap, highly sensitive and highly specific. However, as the appearance of specific antibodies in the body takes some time, the results of tests based on immunoassay remain positive for several weeks after the delayed episode of acute community-acquired lower respiratory tract infection, so they have diagnostic value only in the presence of clinical manifestations of the disease. The genetic approach allows the detection of infectious agents in the early stages of the disease, when serological and immunological methods are ineffective. Tests based on nucleic acid amplification, including PCR, have also become increasingly common recently. These methods should be used for the diagnosis of atypical pathogens and respiratory viruses, because their cultivation in culture is difficult. Sequencing and mass spectrometry methods are being actively developed, but there are limitations that prevent their use in everyday clinical practice. So the combination of microbiological approach with molecular diagnostic methods is the most optimal for the identification of the causative agent of acute community-acquired lower respiratory tract infections and the use of targeted etiotropic treatment. Key words: acute community-acquired lower respiratory tract infections, etiological diagnosis, microbiological, serological, immunological, molecular genetic methods, ICA, PCR, sequencing, mass spectrometry. Ukr. Pulmonol. J. 2021;29(3):58–65

Techno-innovations and molecular methods for diagnosis of COVID-19: Updates from India

The rapid diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection relies on the availability of a robust method. The demand for SARS-CoV-2 diagnostic kits and other desirable consumables for timely diagnosis have increased drastically after the current pandemic. The cumulative efforts by scientists, funding agencies, regulatory authorities, and corporates have accelerated the process of kit development. Worldwide, rapid molecular techno progression to characterize the biology of any pathogen witnessed during this pandemic faster than ever. Moreover, a paradigm shift from real-time polymerase chain reaction technique to highly sensitive novel CRISPR technology has been observed. India, second most populous country actively leveraged molecular technologies for rapid diagnosis of SARS-CoV-2. This paper focuses on paradigm shift in the methods for diagnosis of SARS-CoV-2 infection with a focus on molecular innovations carried out in India indigenously.

&lt;i&gt;In situ&lt;/i&gt;&amp;nbsp;DESI-MSI Lipidomic Profiles of Mucosal Margin of Oral Squamous Cell Carcinoma

Purpose: The optimal safe margin distance of oral squamous cell carcinoma (OSCC) is still controversial. In this article, we apply a cost-effective molecular diagnosis method at the lipidomic level for determining the safe surgical-resection distance of OSCC by desorption electrospray ionization mass spectrometry imaging (DESI-MSI). Methods: A total of 22 participants (each with a matching tumor and continuous mucosal margin tissues) were recruited to the study. All specimens were cut into 10-μm frozen cryosections and scanned by DESI-MSI in the negative ion mode. By overlaying the mass spectrometry image with the H&E staining image, we first precisely extracted the mass spectra of those pixels across the diagnosed tumor and normal regions which served as the training and validation datasets. A Lasso regression model was developed and externally validated for distinguishing the tumor and normal region. A set of feature ions which gradually decreased from tumor to normal tissue was highlighted by assigning them higher weight coefficients during the Lasso model development. The safe surgical-resection distance of OSCC on frozen sections was measured by using this developed molecular diagnostic model for clinical reference. Meanwhile, the genetic marker (p53) of the surgical-margin was used to verify the safe surgical-resection distance determined by DESI-MSI. Results: By leave-one-out validation, the Lasso model can achieve 88.6% accuracy. The Lasso model selected 179 features from an initially collected 22,955 ions across 7,243 pixels for distinguishing tumor and normal tissues. We further screened these 179 characteristic ions, which tend to decrease gradually along with the increase in distance from tumor to normal tissue. Finally, 14 decreasing characteristic ions were tentatively assigned as fatty acids and selected as potential markers for determining the safe surgical-resection distance. These 14 lipid ions gradually decrease from tumor to normal tissue and can be used to accurately distinguish tumors from normal tissues. The spatial segmentation results based on our diagnostic model can not only clearly delineate the tumor and normal tissue but also distinguish the different status of surgical margins. Meanwhile, the safe surgical-resection distance of OSCC on frozen sections can also be accurately measured by using the developed diagnostic model. Moreover, the genetic marker of the OSCC margin (p53) validated that the safe surgical-margin boundary determined by the DESI-MSI and p53 is highly consistent. Conclusions: A diagnostic model was successfully constructed by combining DESI-MSI with the Lasso regression, which could accurately measure the safe margin distance of OSCC on frozen sections. It provides a theoretical basis for a personalized evaluation of safe margin distance of OSCC. Funding Statement: This study was supported by Jiangsu Provincial Key Medical Discipline (since 2017), Nanjing Municipal Key Medical Laboratory Constructional Project Funding (Since 2016), National Natural Science Foundation of China (No.81772880, 81702680). Declaration of Interests: The authors have no competing interests. Ethics Approval Statement: This study was approved by the medical ethics committee of the Nanjing Stomatology Hospital, Medical School of Nanjing University. All participants signed the informed consent forms.

Challenges and Advances in Molecular Diagnosis of Myopathies and Dystrophies in Perspective of Their Use in Developing Countries: Past, Present, and Future.

Proper diagnosis is the first and most critical step for effective identification and treatment of myopathy and dystrophy disorders. Although various histochemical and biochemical studies have paved the way for efficient testing of these disorders, they are insufficient for accurate diagnosis. To overcome this, the diagnostic procedure has now shifted more toward the "genetic first approach," with the remarkable role played by various genetic and molecular techniques. In developing countries, successful diagnosis of such disorders is affected by the shortage of hospitals, poor lab setup, limited diagnostic methods, and unavailability of technical expertise. As a major population living in developing countries faces such inadequate healthcare facilities, there has always been a need for identifying effective diagnostic techniques that could identify genetic alterations more prone in such regions. This article reviews studies done in the last few years that primarily use nonsequencing-based molecular diagnosis methods to identify myopathy- and dystrophy-specific gene alterations and thus could equally hold potential for screening key genetic alterations reported in certain regions in developing countries. Further, this review deals with new emerging sequencing and next-generation sequencing (NGS)-based approach and their potential in providing an adequate diagnosis. This study promotes nonsequencing-based molecular methods to be an effective method for early-stage diagnosis and management of myopathies and dystrophies in developing countries and suggests the high importance of emerging NGS methods in proper diagnosis and identifying new players in neuromuscular disorders.

Comparative evaluation of Conventional and Molecular methods in diagnosis of Tuberculous Meningoencephalitis

Background: Over 1.2 million cases of bacterial meningitis are estimated to occur worldwide each year. World Health Organization (WHO) estimated 9 million people developed tuberculosis in 2013, and 1.5 million died. Tuberculous meningitis (TBM) is still one of the common infections of central nervous system (CNS) and poses significant diagnostic and management challenges, more so in the developing world. The main reason for the spread of tuberculosis is poverty, with resulting homelessness, overcrowding, malnutrition, HIV, excessive alcohol use, diabetes and breakdown of public health infrastructure. Materials and Methods: CSF samples were collected asceptically and processed with an aim to identify and isolate Mycobacterium tuberculosis from clinically suspected cases of chronic meningoencephalitis and compare their conventional and molecular methods of diagnosis. Results: The study group included 197 patients clinically diagnosed as meningoencephalitis. Out of which, 117 had features of chronic meningoencephalitis and were subjected to Z-N staining, culture on LJ media and CBNAAT testing. From 117 cases, 21 cases were AFB positive, 20 cases culture positive and 36 cases were positive for Mycobacterium tuberculosis. Conclusion: CBNAAT (molecular testing) is a better diagnostic tool for diagnosing tuberculous meningitis. Keywords: Meningoencephalitis, Mycobacterium tuberculosis, CBNAAT.